Key Concepts:

Alpha-helix Structural Motif (Interactively
view a molecule in this section!)

Heme Group (Interactively view a molecule
in this section!)

Conformational Changes (Note: This section
contains an animation.)

Spectroscopy and the Color of Blood

Effect of CO2 and H+ on O2
Binding

Integration of Physiological and
Chemical Views

Introduction to the Chemistry and Physiology of
Blood

Our bodies consist of cells that are organized
into many specialized organs and tissues to perform a variety of
functions. Our stomachs digest food so that the nutrients
contained in the food can be distributed to the rest of the body.
Our lungs take in the oxygen needed by the body from the air and
release carbon dioxide as a waste product. Our muscles allow the
body to move. Our brains coordinate all of these (and many other)
activities of the body. These processes are based upon many
different chemical reactions, and the sum total of the chemical
reactions in the body is known as the body's metabolism.
The metabolism includes the reactions needed for normal everyday
activities such as eating, sleeping, and studying. When we
exercise, the metabolism increases to allow our body to cope with
the increased demands and stress of exercising. All of our
specialized body parts are united by their fundamental need for a
particular chemical environment that will enable the body's
metabolic reactions. This environment must include a supply of
nutrients (e.g., sugars and vitamins, to supply the building
blocks for cells and enable biochemical reactions) and oxygen (to
provide energy for the body; the process of using oxygen to make
the body's energy supply is described in the Chem 152 tutorial,
"Energy
for the Body: Oxidative Phosphorylation"), and the
ability to eliminate the waste products of the body's metabolic
activities. This environment is provided by bathing our body's
cells in blood.

Blood is part of the body's circulatory system,
and thus is continually being pumped through our bodies as long
as we are alive. The blood distributes oxygen and nutrients to
the many different cells in the body, carries CO2
generated by the cells to the lungs for exhalation, and carries
other waste products to the kidneys and liver for processing and
elimination. Many finely tuned chemical processes occur in the
blood to allow the blood to carry out all of these functions and
provide for the needs of the body. The tutorials in Chem 151 and
152 will describe several of these vital chemical processes, such
as the release of iron in controlled amounts to the blood ("Iron
Use and Storage in the Body: Ferritin and Molecular
Representations" tutorial), removal of waste products
from the blood ("Maintaining
the Body's Chemistry: Dialysis in the Kidneys"
tutorial), and the regulation of the levels of CO2 and
H+ to control the pH of the blood ("Blood,
Sweat, and Buffers: pH Regulation During Exercise"
tutorial). In this tutorial, we will study one of the most
important functions of blood, the transport of oxygen from the
lungs to the other cells of the body (e.g., muscle cells) that
perform metabolic functions.

Oxygen Transport via Metal Complexes

An adult at rest consumes the equivalent of 250
ml of pure oxygen per minute. This oxygen is used to provide
energy for all the tissues and organs of the body, even when the
body is at rest. The body's oxygen needs increase dramatically
during exercise or other strenuous activities. The oxygen is
carried in the blood from the lungs to the tissues where it is
consumed. However, only about 1.5% of the oxygen transported in
the blood is dissolved directly in the blood plasma. Transporting
the large amount of oxygen required by the body, and allowing it
to leave the blood when it reaches the tissues that demand the
most oxygen, require a more sophisticated mechanism than simply
dissolving the gas in the blood. To meet this challenge, the body
is equipped with a finely-tuned transport system that centers on
the metal complex heme.

Metal Complexes in the Body

The ability of metal ions to coordinate with (bind) and then
release ligands in some processes, and to oxidize and reduce in
other processes makes them ideal for use in biological systems.
The most common metal used in the body is iron, and it plays a
central role in almost all living cells. For example, iron
complexes are used in the transport of oxygen in the blood and
tissues.

Metal-ion complexes consist of a metal ion that is bonded via
"coordinate-covalent bonds" (Figure 1) to a small
number of anions or neutral molecules called ligands. For example
the ammonia (NH3) ligand used in this experiment is a
monodentate ligand; i.e., each monodentate ligand in a
metal-ion complex possesses a single electron-pair-donor atom and
occupies only one site in the coordination sphere of a metal ion.
Some ligands have two or more electron-pair-donor atoms that can
simultaneously coordinate to a metal ion and occupy two or more
coordination sites; these ligands are called polydentate ligands.
They are also known as chelating agents (from the Greek word
meaning "claw"), because they appear to grasp the metal
ion between two or more electron-pair-donor atoms. The
coordination number for a metal refers to the total number of
occupied coordination sites around the central metal ion (i.e.,
the total number of metal-ligand bonds in the complex).

Figure 1

You have already learned that a covalent bond forms
when electrons are shared between atoms. A coordinate-covalent
bond (represented by a green arrow in this
diagram) forms when both of the shared electrons come
from the same atom, called the donor atom
(blue).

An anion or molecule containing the donor atom is
known as a ligand. The top illustration
shows a coordinate-covalent bond between a metal ion
(e.g., Fe, shown in red) and a monodentate
ligand (a ligand that contains only one
electron-pair-donor atom, shown in light blue). The
bottom illustration shows a metal ion with
coordinate-covalent bonds to a bidentate ligand
(a ligand that contains two donor atoms simultaneously
coordinated to the metal ion, shown in yellow).

Oxygen-Carrying Protein in the Blood: Hemoglobin

Hemoglobin is the protein that transports oxygen (O2)
in human blood from the lungs to the tissues of the body.
Proteins are formed by the linking of amino acids into
polypeptide chains. An individual amino acid in a protein is
known as a "residue." The arrangement and interactions
of the amino-acid residues within the protein determine the
protein's shape and contribute substantially to its function.
Hemoglobin is a globular protein (i.e., folded into a compact,
nearly spherical shape) and consists of four subunits, as shown
in Figure 2. Each protein subunit is an individual molecule that
joins to its neighboring subunits through intermolecular
interactions. (These subunits are also known as peptide
chains. You will learn more about the nature of amino
acids and peptide subunits in the tutorial entitled, "Iron
Use and Storage in the Body: Ferritin and Molecular
Representations".)

Figure 2

This is a molecular model of hemoglobin with the
subunits displayed in the ribbon representation. A ribbon
representation traces the backbone atoms of a protein and
is often used to represent its three-dimensional
structure. The four heme groups are displayed in the
ball-and-stick representation.

Note: The coordinates for the
hemoglobin protein (in this and subsequent molecular
representations of all or part of the protein) were
determined using x-ray crystallography, and the image was
rendered using SwissPDB Viewer and POV-Ray (see References).

Note: To
view the molecule interactively, please use Jmol,
and click on the button to the left.

To understand the oxygen-binding properties of hemoglobin, we
will focus briefly on the structure of the protein and the metal
complexes embedded in it.

The Protein Subunit

Each subunit in Figure 2 contains regions with a coiled shape;
many of the amino acids that make up the polypeptide chain
interact to form this particular structure, called an alpha
helix. In an alpha helix (Figure 3), each amino acid is
"hydrogen-bonded" to the amino acid that is four
residues ahead of it in the chain. In hemoglobin, the
hydrogen-bonding interaction occurs between the H of an -NH group
and the O of a -CO group of the polypeptide backbone chain; the
amino-acid side chains extend outward from the backbone of the
helix. Approximately 75% of the amino-acid composition of
hemoglobin adopts an alpha-helical structure. Another common
structural motif is the beta-pleated sheet, in which amino acids
line up in straight parallel rows.

Figure 3

This is a molecular model of the
alpha-helix structure in a subunit of hemoglobin. The
blue strands are a ribbon representation to emphasize the
helical structure. The green dotted lines show the
hydrogen bonding between the -NH and -CO functional
groups.

Note: To view the
molecule interactively, please use Jmol,
and click on the button above.

Please click on the pink button
above to view a QuickTime movie showing a
rotation of the alpha-helix structure shown in
Figure 3.

Click the blue button below to
download QuickTime
4.0 to view the movie.

The Heme Group

In hemoglobin, each subunit contains a heme group, which is
displayed using the ball-and-stick representation in Figure 2.
Each heme group contains an iron atom that is able to bind to one
oxygen (O2) molecule. Therefore, each hemoglobin
protein can bind four oxygen molecules.

One of the most important classes of chelating agents in
nature are the porphyrins. A porphyrin molecule can coordinate to
a metal using the four nitrogen atoms as electron-pair donors,
and hence is a polydentate ligand (see Figure 1). Heme is a
porphyrin that is coordinated with Fe(II) and is shown in Figure
4.

Figure 4

On the left is a three-dimensional
molecular model of heme coordinated to the histidine
residue (a monodentate ligand, see Figure 1) of the
hemoglobin protein. On the right is a two-dimensional
drawing of heme coordinated to the histidine residue,
which is part of the hemoglobin protein. In
this figure, the protein is deoxygenated; i.e., there
is no oxygen molecule bound to the heme group.

Note: The
coordinate-covalent bonds between the central iron atom
and the nitrogens from the porphyrin are gold; the
coordinate-covalent bond between the central iron atom
and the histidine residue is green. In the
three-dimensional model, the carbon atoms are
are gray, the iron atom is dark red,
the nitrogen atoms are dark blue, and the oxygen atoms
are light red. The rest of the hemoglobin
protein is purple.

Note: To
view the molecule interactively, please use Jmol,
and click on the button to the left.

In the body, the iron in the heme is coordinated to the four
nitrogen atoms of the porphyrin and also to a nitrogen atom from
a histidine residue (one of the amino-acid residues in
hemoglobin) of the hemoglobin protein (see Figure 4). The sixth
position (coordination site) around the iron of the heme is
occupied by O2 when the hemoglobin protein is
oxygenated.

Questions on the Oxygen-Carrying Protein in the Blood:
Hemoglobin

One peptide subunit in hemoglobin contains 141 amino-acid
residues. If the subunit were stretched out, it would
measure approximately 49 nm in length. However, the
longest dimension of the subunit in hemoglobin is only
about 5 nm. Briefly, explain how alpha helices may help
account for this difference in length.

What is the coordination number of Fe in the oxygenated
heme group? Briefly, justify your answer by describing
the ligands to which Fe is coordinated.

Conformational Changes Upon Binding of Oxygen

Careful examination of Figure 4 shows that the heme group is
nonplanar when it is not bound to oxygen; the iron atom is pulled
out of the plane of the porphyrin, toward the histidine residue
to which it is attached. This nonplanar configuration is
characteristic of the deoxygenated heme group, and is commonly
referred to as a "domed" shape. The valence electrons
in the atoms surrounding iron in the heme group and the valence
electrons in the histidine residue form "clouds" of
electron density. (Electron density refers to the probability of
finding an electron in a region of space.) Because electrons
repel one another, the regions occupied by the valence electrons
in the heme group and the histidine residue are pushed apart.
Hence, the porphyrin adopts the domed (nonplanar) configuration
and the Fe is out of the plane of the porphyrin ring (Figure 5,
left). However, when the Fe in the heme group binds to an oxygen
molecule, the porphyrin ring adopts a planar configuration and
hence the Fe lies in the plane of the porphyrin ring (Figure 5,
right).

Figure 5

On the left is a schematic diagram showing
representations of electron-density clouds of the
deoxygenated heme group (pink) and the attached histidine
residue (light blue). These regions of electron density
push one another apart, and the iron atom in the center
is drawn out of the plane. (The nonplanar shape of the
heme group is represented by the bent line.)

On the right is a schematic diagram showing
representations of electron-density clouds of the
oxygenated heme group (pink), the attached histidine
residue (light blue), and the attached oxygen molecule
(gray). The oxygenated heme assumes a planar
configuration, and the central iron atom occupies a space
in the plane of the heme group (depicted by a straight
red line).

The shape change in the heme group has important implications
for the rest of the hemoglobin protein, as well. When the iron
atom moves into the porphyrin plane upon oxygenation, the
histidine residue to which the iron atom is attached is drawn
closer to the heme group. This movement of the histidine residue
then shifts the position of other amino acids that are near the
histidine (Figure 6). When the amino acids in a protein are
shifted in this manner (by the oxygenation of one of the heme
groups in the protein), the structure of the interfaces between
the four subunits is altered. Hence, when a single heme group in
the hemoglobin protein becomes oxygenated, the whole protein
changes its shape. In the new shape, it is easier for the other
three heme groups to become oxygenated. Thus, the binding of one
molecule of O2 to hemoglobin enhances the ability of
hemoglobin to bind more O2 molecules. This property of
hemoglobin is known as "cooperative binding."

Figure 6

This figure shows the heme group and a portion of the
hemoglobin protein that is directly attached to the heme.
When hemoglobin is deoxygenated (left), the heme group
adopts a domed configuration. When hemoglobin is
oxygenated (right), the heme group adopts a planar
configuration. As shown in the figure, the conformational
change in the heme group causes the protein to change its
conformation, as well.

Please click on the pink button below to view a
QuickTime movie showing how the amino acid residues near
the heme group in hemoglobin shift as the heme group
converts between the nonplanar (domed) and the planar
conformation by binding and releasing a molecule of O2.
Click the blue button below to download QuickTime 4.0
to view the movie.

Questions on Conformational Changes Upon Binding of Oxygen:

Explain, in terms of electron repulsion, why the heme
group adopts a nonplanar (domed) configuration upon
deoxygenation.

Explain how a change in the heme group configuration causes the entire
hemoglobin subunit to change shape.

Spectroscopy and the Color of Blood

The changes that occur in blood upon oxygenation and
deoxygenation are visible not only at the microscopic level, as
detailed above, but also at the macroscopic level. Clinicians
have long noted that blood in the systemic arteries (traveling
from the heart to the oxygen-using cells of the body) is
red-colored, while blood in the systemic veins (traveling from
the oxygen-using cells back to the heart) is blue-colored (see
Figure 7). The blood in the systemic arteries is oxygen-rich;
this blood has just traveled from the lungs (where it picked up
oxygen inhaled from the air) to the heart, and then is pumped
throughout the body to deliver its oxygen to the body's cells.
The blood in the systemic veins, on the other hand, is
oxygen-poor; it has unloaded its oxygen to the body's cells
(exchanging the O2 for CO2, as described
below), and must now return to the lungs to replenish the supply
of oxygen. Hence, a simple macroscopic observation, i.e., noting
the color of the blood, can tell us whether the blood is
oxygenated or deoxygenated.

What causes this color change in the blood? We know that the
shape of the heme group and the hemoglobin protein change,
depending on whether hemoglobin is oxygenated or deoxygenated.
The two conformations must have different light-absorbing
properties. The oxygenated conformation of hemoglobin must absorb
light in the blue-green range, and reflect red light, to account
for the red appearance of oxygenated blood. The deoxygenated
conformation of hemoglobin must absorb light in the orange range,
and reflect blue light, to account for the bluish appearance of
deoxygenated blood. We could use a spectrophotometer to examine a dilute solution of
blood and determine the wavelength of light absorbed by each
conformation. For an approximate prediction of the wavelength of
light absorbed and for the colors of light absorbed for a given complementary color, a table such as Table 1 in the introduction to
the Experiment ("Relations Between Electronic Transition
Energy and Color") could be used.

Questions on Spectroscopy and the Color of Blood

Propose an explanation for why the change in heme group conformation
results in a color change.

A researcher prepares two solutions of deoxygenated
hemoglobin. One solution is ten times as concentrated as
the other solution. The researcher then obtains
absorption spectra for the two solutions.

Do you expect the wavelength of maximum absorption (lmax) to be the same
or different for the two solutions? If lmax is different for
the two solutions, indicate which solution will have
a higher lmax.
Briefly, explain your reasoning.

Do you expect the absorbance (A) at lmax to be the same
or different for the two solutions? If the absorbance
is different for the two solutions, indicate which
solution will have a higher absorbance. Briefly,
explain your reasoning.

The Effect of CO2 and H+ on O2
Binding

In 1904, Christian Bohr discovered that increased
concentrations of CO2 and H+ promote the
release of O2 from hemoglobin in the blood. This
phenomenon, known as the Bohr effect, is a highly adaptive
feature of the body's blood-gas exchange mechanism. The blood
that is pumped from the heart to the body tissues and organs
(other than the lungs) is rich in oxygen (Figure 7). These
tissues require oxygen for their metabolic activities (e.g.,
muscle contraction). Hence, it is necessary for oxygen to remain
bound to hemoglobin as the blood travels through the arteries (so
that it can be carried to the tissues), but be easily removable
when the blood passes through the capillaries feeding the body
tissues. CO2 and H+ are produced from
metabolic activities of the body, and so the concentration of
these species is high in the metabolically active tissues of the
body. Thus, the tissues that perform the most metabolic activity
(and therefore require the largest amount of O2)
produce large quantities of CO2 and H+,
facilitating the release of O2 from the blood where
the O2 is most needed. In the lungs, the reverse
effect occurs: high concentrations of O2 cause the
release of CO2 from hemoglobin.

Blood rich in carbon dioxide is pumped from the
heart into the lungs through the pulmonary
arteries. (Arteries are blood vessels carrying
blood away from the heart; veins are blood
vessels carrying blood to the heart.)

In the lungs, CO2 in the blood is
exchanged for O2.

The oxygen-rich blood is carried back to the
heart through the pulmonary veins.

This oxygen-rich blood is then pumped from the
heart to the many tissues and organs of the body,
through the systemic arteries.

In the tissues, the arteries narrow to tiny
capillaries. Here, O2 in the blood is
exchanged for CO2.

The capillaries widen into the systemic veins,
which carry the carbon-dioxide-rich blood back to
the heart.

Figure 7

This is a schematic diagram of the flow of blood
through the circulatory system, showing the sites of O2/CO2
exchange in the body.

Note: The components of this diagram
are not drawn to scale.

How do CO2 and H+ promote the release of
O2 from hemoglobin? These species help form
interactions between amino-acid residues at the interfaces of the
four subunits in hemoglobin. These interactions are called "salt bridges,"
because they are between positively-charged and negatively-charged amino-acid
residues on different subunits of the same protein (Figure 8). When
"salt bridges" form, the subunits are held in a position that
"tugs on" the histidine that is attached to the heme iron. (See Figure
5.) This favors the domed configuration, which is the deoxygenated form of
hemoglobin.

Figure 8

On the left is a schematic diagram of the interface of
two subunits of the deoxygenated hemoglobin protein. In
the presence of CO2 and H+ (e.g.,
in the muscles), charged groups are formed on the amino
acid residues lining the subunit interface. These charged
groups are held together by ionic interactions, forming
"salt bridges" between the two subunits, and
stabilizing the deoxygenated form of hemoglobin.

When blood passes through the alveolar capillaries of
the lungs, CO2 and H+ are removed
from the hemoglobin, and the oxygenated configuration is
favored (right).

Note: The components of this diagram
are not drawn to scale.

When the concentration of protons (H+) is low (pH
9), positive charges do not form on the residues at the subunit
interfaces, so the salt bridges cannot form (right image in
Figure 8). However, at pH 7, histidine residues at the subunit
interfaces (not the histidine residues that bind the heme groups)
can accept an additional proton (H+), and hence become
positively charged (Equation 1). When salt bridges form by the
interaction of these interfacial histidine residues and nearby
negatively-charged amino-acid residues, the deoxygenated
hemoglobin structure is favored, and oxygen is released (left
image in Figure 8).

(1)

The number of negatively-charged residues in the salt bridges
is increased in the presence of carbon dioxide. CO2
binds to the amino (-NH2) group of certain amino acid
residues at the subunit interfaces to produce a
negatively-charged group (-NHCOO-) on the residue
(Equation 2). This negatively-charged group can form salt bridges
with the positive charges on the protonated histidines described
above. The H+ produced by Equation 2 can also be
accepted by histidine residues at the subunit interfaces (via
Equation 1).

(2)

Thus, hemoglobin's biological function is regulated by the
changing of the overall protein structure. This structure is
altered by the binding or releasing of CO2 and H+
to the interfaces of the subunits in hemoglobin (Figure 8).

Questions on the Bohr Effect:

Does CO2 bind at the same site on the hemoglobin molecule as
O2? If not, where does CO2 bind?

In muscles, the oxygen released by
hemoglobin is taken up by myoglobin. Myoglobin is a
muscular protein that stores oxygen and allows it to
diffuse throughout the muscle fibers so that it can be
used by the muscle. Myoglobin contains only one subunit
(and thus only one heme group), which is very similar to
one of the subunits of hemoglobin. Would you expect myoglobin to
exhibit the Bohr effect (i.e., would myoglobin
release O2in the presence of
CO2 and H+)? Briefly, explain
your reasoning.

Summary

Blood is an amazing and vitally important part of the body,
because it contains many finely-tuned chemical systems that allow
it to maintain the chemical environment needed for the body's
metabolism. One of the most important functions of blood is
delivering O2 to all parts of the body by the
hemoglobin protein. O2 is carried in the hemoglobin
protein by the heme group. The heme group (a component of the
hemoglobin protein) is a metal complex, with iron as the central
metal atom, that can bind or release molecular oxygen. Both the
hemoglobin protein and the heme group undergo conformational
changes upon oxygenation and deoxygenation. When one heme group
becomes oxygenated, the shape of hemoglobin changes in such a way
as to make it easier for the other three heme groups in the
protein to become oxygenated, as well. This feature helps the
protein to pick up oxygen more efficiently as the blood travels
through the lungs. Hemoglobin also enables the body to eliminate
CO2, which is generated as a waste product, via gas
exchange in the blood (CO2 exchanged for O2
in the lungs, and O2 exchanged for CO2 in
the muscles). The species generated as waste by the
oxygen-consuming cells actually help to promote the release of O2
from hemoglobin when it is most needed by the cells. Hence,
hemoglobin is a beautiful example of the finely tuned chemical
systems that enable the blood to distribute necessary molecules
to cells throughout the body, and remove waste products from
those cells.

Jmol Files

To view the molecules interactively, please use Jmol.
To download the pdb files for viewing and rotating the molecules
shown above, please click on the appropriate name below or on the
"interactive" button below each molecular-model figure
in the text.

Additional Links:

For additional information on hemoglobin, see the hemoglobin
tutorial by Eric Martz of the University of
Massachusetts.

Note: You will use Jmol to view this
tutorial. More information regarding Jmol can be found here.

To learn about porphyrin and other chelating
agents, see this "Chemical of the Week"
site from the University of Wisconsin.

Another porphyrin molecule, similar to heme but
containing Mg rather than Fe as the central atom, is chlorophyll.
You can learn about the structure , function, and
spectroscopy of chlorophyll
from this site. (Note: You will be
studying chlorophyll in lab at the beginning of Chemistry
152.)

Acknowledgements:

The authors thank Greg Noelken for creating the Jmol script files.
They also wish to thank Dewey Holten, Michelle Gilbertson, Jody
Proctor and Carolyn Herman for many helpful suggestions in the writing of this
tutorial.

The development of this tutorial was supported by a grant from
the Howard Hughes Medical Institute, through the Undergraduate
Biological Sciences Education program, Grant HHMI# 71199-502008
to Washington University.